Reconstitution and characterization of the polynuclear iron-sulfur cluster in pyruvate formate-lyase-activating enzyme. Molecular properties of the holoenzyme form.
The glycyl radical (Gly-734) contained in the active form of pyruvate formate-lyase (PFL) of Escherichia coli is generated by the S-adenosylmethionine-dependent pyruvate formate-lyase-activating enzyme (PFL activase). A 5'-deoxyadenosyl radical intermediate produced by the activase has been suggested as the species that abstracts the pro-S hydrogen of the glycine 734 residue in PFL (Frey, M., Rothe, M., Wagner, A. F. V., and Knappe, J. (1994) J. Biol. Chem. 269, 12432-12437). To enable mechanistic investigations of this system we have worked out a convenient large scale preparation of functionally competent PFL activase from its apoform. The previously inferred metallic cofactor was identified as redox-interconvertible polynuclear iron-sulfur cluster, most probably of the [4Fe-4S] type, according to UV-visible and EPR spectroscopic information. Cys --> Ser replacements by site-directed mutagenesis determined Cys-29, Cys-33, and Cys-36 to be essential to yield active holoenzyme. Gel filtration chromatography showed a monomeric structure (28 kDa) for both the apoenzyme and holoenzyme form. The iron-sulfur cluster complement proved to be a prerequisite for effective binding of adenosylmethionine, which induces a characteristic shift of the EPR signal shape of the reduced enzyme form ([4Fe-4S]+) from axial to rhombic symmetry.
Külzer
R
R
Institut für Biologische Chemie, University of Heidelberg, D-69120 Heidelberg, Germany.
Pils
T
T
Kappl
R
R
Hüttermann
J
J
Knappe
J
J
D000123:Acetyltransferases; D000595:Amino Acid Sequence; D001059:Apoproteins / Q000378:metabolism; D017124:Conserved Sequence; D003545:Cysteine / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D004798:Enzymes / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D007501:Iron / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000096:biosynthesis* / Q000235:genetics; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis; D012435:S-Adenosylhomocysteine / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism; D013053:Spectrophotometry; D013440:Sulfides / Q000378:metabolism
D001059:Apoproteins; D004798:Enzymes; D007506:Iron-Sulfur Proteins; D008024:Ligands; D011994:Recombinant Proteins; D013440:Sulfides; D012436:S-Adenosylmethionine; D012435:S-Adenosylhomocysteine; D007501:Iron; D000123:Acetyltransferases; C040285:pyruvate formate-lyase activating enzyme; D003545:Cysteine